Abstract The proposed project integrates groups from academia and industry to develop and perform initial testing of a novel platform for the potential eradication of HIV-1 by endowing the patient's own cells with stable resistance to HIV infection. To safely and effectively achieve such resistant cells, we propose to deploy a process call genome editing to either (i) perform targeted insertion of anti-HIV gene sequences, and/or permanently disrupt HIV relevant gene targets, directly in the genome of the patient's hematopoietic stem/progenitor cells (HSPC). Genome editing is achieved by invoking the cell's own natural DNA repair pathways to heal a double-strand break (DSB) introduced in vivo at the target locus by an engineered DNA cleavage enzyme called a zinc finger nuclease (ZFN). This technology has recently entered the clinic in subjects with HIV, where the safety, tolerability and early evidence of genetic protection from HIV infection has been observed following an infusion of autologous CCR5 gene edited CD4+ T cells. While CCR5 is a validated target for development of HIV therapeutics, disruption of this gene in isolation will not confer resistance to dual or X4-tropic HIV isolates. To expand the potential application of genome edited HSPCs to achieve a potential cure for HIV our strategy is perform the genetic addition of anti-HIV gene products to a defined location in the stem cell genome. Specifically, we propose to develop targeted nucleases to enable the in situ editing of endogenous human restriction factors as well develop methods to achieve the site-specific addition of anti-HIV genes in the critical long term hematopoietic stem cell (LT-HSC). The latter has recently been identified as a key area for optimization of genome editing strategies in these promising therapeutic stem cells.